The invention is directed to purified and isolated human polypeptides having kinase function, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, the use of such polypeptides and fragmented peptides in phosphorylation reactions and as molecular weight markers, the use of such polypeptides and fragmented peptides as controls for peptide fragmentation, the use of such polypeptides in screening assays, and kits comprising these reagents.
The eukaryotic protein kinases make up a large and rapidly expanding family of proteins related on the basis of homologous catalytic domains. Spurred by the development of gene cloning and sequencing methodologies, distinct protein kinase genes have been identified from a wide selection of invertebrates and lower eukaryotes, including Drosophila, Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium, and budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. Homologous genes have also been identified in higher plants. Protein kinases, however, are not limited to the eukaryotes. Enzyme activities have been well documented in prokaryotes, but the prokaryotic protein kinase genes are not obviously homologous to those of the eukaryotes. Because protein kinases are useful biochemical reagents, there is a need in the art for the continued discovery of unique members of the protein kinase family.
In addition, the discovery and identification of proteins are at the forefront of modern molecular biology and biochemistry. The identification of the primary structure, or sequence, of a sample protein is the culmination of an arduous process of experimentation. In order to identify an unknown sample protein, the investigator can rely upon comparison of the unknown sample protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, and mass spectrometry.
Comparison of an unknown protein sample to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein sample (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)). Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein samples (New England Biolabs Inc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown sample protein to allow an accurate estimation of apparent molecular weight.
The difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means (A. L. Lehninger, Biochemistry 106-108 (Worth Books, 2d ed. 1981)). Chemical fragmentation can be achieved by incubation of a protein with a chemical, such as cyanogen bromide, which leads to cleavage of the peptide bond on the carboxyl side of methionine residues (E. Gross, Methods in Enz. 11:238-255, 1967). Enzymatic fragmentation of a protein can be achieved by incubation of a protein with a protease that cleaves at multiple amino acid residues (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977). Enzymatic fragmentation of a protein can also be achieved by incubation of a protein with a protease, such as Achromobacter protease I (F. Sakiyama and A. Nakata, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim. Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta 660:51-55, 1981), which leads to cleavage of the peptide bond on the carboxyl side of lysine residues. The molecular weights of the fragmented peptides can cover a large range of molecular weights and the peptides can be numerous. Variations in the degree of fragmentation can also be accomplished (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977).
The unique nature of the composition of a protein with regard to its specific amino acid constituents results in a unique positioning of cleavage sites within the protein. Specific fragmentation of a protein by chemical or enzymatic cleavage results in a unique xe2x80x9cpeptide fingerprintxe2x80x9d (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980). Consequently, cleavage at specific sites results in reproducible fragmentation of a given protein into peptides of precise molecular weights. Furthermore, these peptides possess unique charge characteristics that determine the isoelectric pH of the peptide. These unique characteristics can be exploited using a variety of electrophoretic and other techniques (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)).
When a peptide fingerprint of an unknown protein is obtained, this can be compared to a database of known proteins to assist in the identification of the unknown protein (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; B. Thiede et al., Electrophoresis 1996, 17:588-599, 1996). A variety of computer software programs are accessible via the Internet to the skilled artisan for the facilitation of such comparisons, such as MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.emblheiedelberg.de...deSearch/FR_PeptideSearchForm.html), and ProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein databases to assist in the elucidation of the identity of the sample protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in the determination of the number of fragmented peptides and the precise molecular weight of those peptides should result in enhanced success in the identification of unknown proteins.
Fragmentation of proteins is further employed for the production of fragments for amino acid composition analysis and protein sequencing (P. Matsudiara, J. Biol. Chem. 262:10035-10038, 1987; C. Eckerskom et al., Electrophoresis 1988, 9:830-838, 1988), particularly the production of fragments from proteins with a xe2x80x9cblockedxe2x80x9d N-terminus. In addition, fragmentation of proteins can be used in the preparation of peptides for mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad Sci. USA 90:5011-5015, 1993; B. Thiede et al., Electrophoresis 1996, 17:588-599, 1996), for immunization, for affinity selection (R. A. Brown, U.S. Pat. No.5,151,412), for determination of modification sites (e.g. phosphorylation), for generation of active biological compounds (T. D. Brock and M. T. Madigan, Biology ofMicroorganisms 300-301 (Prentice Hall, 6d ed. 1991)), and for differentiation of homologous proteins (M. Brown et al., J. Gen. Virol. 50:309-316, 1980).
In view of the continuing interest in protein research and the elucidation of protein structure and properties, there exists a need in the art for polypeptides having kinase function or suitable for use in peptide fragmentation studies and in molecular weight measurements.
The invention aids in fulfilling these needs in the art. The invention encompasses an isolated human nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15 and an isolated human nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 14, or 16. The invention also encompasses nucleic acid molecules complementary to these sequences. As such, the invention includes double-stranded nucleic acid molecules comprising the DNA sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15 and isolated nucleic acid molecules encoding the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 14 or 16. Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention. These molecules can be used to detect both single-stranded and double-stranded RNA and DNA variants encompassed by the invention. A double-stranded DNA probe allows the detection of nucleic acid molecules equivalent to either strand of the nucleic acid molecule. Isolated nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising the DNA sequence of SEQ ID NO:1, 2,3, 4, 5,6, 13, or 15, or an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 14, or 16 are within the invention. A preferred set of hybridization conditions are those of moderate stringency: in 50% formamide and 6xc3x97SSC, at 42xc2x0 C. with washing conditions of 60xc2x0 C., 0.5xc3x97SSC, 0.1% SDS.
The invention further encompasses isolated nucleic acid molecules derived by in vitro mutagenesis from SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or 15. In vitro mutagenesis would include numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis. The invention also encompasses isolated nucleic acid molecules degenerate from SEQ ID NO:1, 2, 3, 4, 5, or 6 (and the resulting amino acid sequence) as a result of the genetic code, isolated nucleic acid molecules that are allelic variants of human DNA of the invention, or a species homolog of DNA of the invention. The invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors. In addition, the invention encompasses methods of using the nucleic acid noted above in assays to identify chromosomes, map human genes, and study tumors.
The invention also encompasses isolated polypeptides encoded by these nucleic acid molecules, including isolated polypeptides having a molecular weights as determined by SDS-PAGE, isolated polypeptides in non-glycosylated form, and fragments thereof. The invention further includes synthetic polypeptides encoded by these nucleic acid molecules. Peptides and fragments of these polypeptides, however derived, are also part of the invention and may be produced by any standard means, from chemical, enzymatic, recombinant, or synthetic methods. Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are encompassed by the invention. The invention further encompasses methods for the production of polypeptides having kinase functions including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium. Especially, the expression of polypeptides having kinase functions in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
In general, the polypeptides of the invention having kinase function can be used to phosphorylate target proteins and to radiolabeled target proteins with 32P. In addition, the polypeptides of the invention having kinase function can be used to identify proteins having a phosphate activity.
In addition, assays utilizing polypeptides having kinase functions to screen for potential inhibitors of activity associated with polypeptide counter-structure molecules, and methods of using polypeptides having kinase functions as therapeutic agents for the treatment of diseases mediated by polypeptide counter-structure molecules are encompassed by the invention. Methods of using polypeptides having kinase functions in the design of inhibitors thereof are also an aspect of the invention. The invention further encompasses use of polypeptides of the invention to screen for agonists and antagonists.
The invention further encompasses the fragmented peptides produced from polypeptides of the invention by chemical or enzymatic treatment. In addition, the polypeptides of the invention and fragmented peptides thereof, wherein at least one of the sites necessary for fragmentation by chemical or enzymatic means has been mutated, are an aspect of the invention.
The invention further includes a method for using these polypeptides and fragmented peptides thereof as molecular weight markers that allow the estimation of the molecular weight of a protein or a fragmented protein sample. The invention also encompasses a method for the visualization of the molecular weight markers of the invention thereof using electrophoresis. The invention further encompasses methods for using the polypeptides of the invention and fragmented peptides thereof as markers, which aid in the determination of the isoelectric point of a sample protein. The invention also encompasses methods for using polypeptides of the invention and fragmented peptides thereof as controls for establishing the extent of fragmentation of a protein sample.
Further encompassed by this invention are kits to aid the determination of molecular weights of a sample protein utilizing polypeptide molecular weight markers of the invention, fragmented peptides thereof, and forms of these polypeptide molecular weight markers, wherein at least one of the sites necessary for fragmentation by chemical or enzymatic means has been mutated.